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Proceedings Paper

Interactive algorithms for rapid enumeration of hybridization signals in individual whole-cell nuclei inside intact-tissue specimens
Author(s): Stephen J. Lockett; Curtis T. Thompson; James C. Mullikin; Damir Sudar; R. Khavari; William Hyun; Daniel Pinkel; Joe W. Gray
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Paper Abstract

Fluorescence in situ hybridization (FISH) is useful for analyzing specific nucleic acid sequences in individual cells. Its application to tissue sections has been limited however because of the difficulties of performing the hybridization and analysis in sections that are thick enough to contain intact nuclei. Recent improvements in FISH permit hybridization with chromosome-specific, centromeric probes throughout 20 micrometers formalin fixed, paraffin- embedded sections, which do contain many intact nuclei. This paper describes software to facilitate analysis of these 3D hybridizations. We have developed two algorithms for analyzing 3D, confocal images of thick sections. One displays 2D, maximum-intensity, projection images through the original 3D image at different angles. When projections are viewed sequentially, the 3D image appears semi-transparent and rotates. The second algorithm allows interactive enumeration of FISH signals. Each signal is marked by the analyst. Then, for each pair of marked signals, a 2D slice image along the line connecting both marked signals and parallel to the z (depth) axis is displayed. From this slice, the analyst decides if the signals are in the same or different nuclei, or if the signals should be rejected because they are in a nucleus truncated by the upper or lower surface of the section. After consideration of all pairs of signals, the algorithm produces a map of the tissue section showing the numbers of signals in each of the intact nucleus. The algorithms enable analysis of small, premalignant and early malignant lesions and infiltrative lesions that cannot be analyzed by other molecular techniques and permit the direct correlation of FISH information with histology/cytology.

Paper Details

Date Published: 23 March 1995
PDF: 7 pages
Proc. SPIE 2412, Three-Dimensional Microscopy: Image Acquisition and Processing II, (23 March 1995); doi: 10.1117/12.205346
Show Author Affiliations
Stephen J. Lockett, Lawrence Berkeley Lab. (United States)
Curtis T. Thompson, Univ. of California/San Francisco (United States)
James C. Mullikin, Lawrence Berkeley Lab. (United States)
Damir Sudar, Lawrence Berkeley Lab. (United States)
R. Khavari, Univ. of California/San Francisco (United States)
William Hyun, Univ. of California/San Francisco (United States)
Daniel Pinkel, Lawrence Berkeley Lab. (United States)
Joe W. Gray, Lawrence Berkeley Lab. (United States)


Published in SPIE Proceedings Vol. 2412:
Three-Dimensional Microscopy: Image Acquisition and Processing II
Tony Wilson; Carol J. Cogswell, Editor(s)

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